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First published online 5 December 2007
doi: 10.1242/dev.012054

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Wnt canonical pathway restricts graded Shh/Gli patterning activity through the regulation of Gli3 expression

¹ Instituto de Biología Molecular de Barcelona, CSIC, Parc Cientific de Barcelona, C/Josep Samitier 1-5, Barcelona 08028, Spain.
² Okazaki Institute for Integrative Biosciences, National Institutes of Natural Sciences, Okazaki 444-8787 Japan.

View larger version (49K): [in this window] [in a new window]   Fig. 1. Dorsally expressed Wnt genes regulate expression of progenitor proteins and result in cell fate changes along the DV axis. (A,B) HH stage 18 chick embryos show that expression of Wnt1 and Wnt3a is restricted to the dorsal-most neural tube. (C-G) Eight hours post-electroporation (8 hours PE) of Wnt1/3a, expression of Pax7 (C) and Pax6 (D) is ventrally expanded. Green shows GFP expression as reporter of ectopic Wnt gene expression (E). Expression of ventral Olig2 (F) and Nkx2.2 (G) is reduced. (H-Q) Twenty-four hours PE, Wnt1/3a causes overgrowth of the electroporated side, expansion of Pax7 (H) and Pax6 (I) expression, loss of intermediate genes Dbx1 (K) and Dbx2 (L), and loss of ventral Olig2 (M) and of Nkx2.2 (N), Foxa2 (O), Nkx6.2 (P) and Nkx6.1 (Q). (J) Green shows GFP expression as a reporter of ectopic Wnt expression. (R-T) Forty-eight hours PE, Wnt1/3a causes phenotype changes on differentiated neurons. (R) Quantitative analysis of Lhx1/5+/Pax2-dI2, Islet1+ dI3 and Lhx1/5+/Pax2+ dI4-dI6/V1 interneurons and of ventral Islet1+ motoneurons (MN) on Wnt1/Wnt3a electroporated versus control pCIG electroporated embryos. (S,T) Double immunofluorescence staining with specific markers for each neuronal population: Lhx1/5+/Pax2-for dI2 (red); Lhx1/5+/Pax2+ for dI4-dI6/V1 (yellow) (S) and Islet1+ for dI3 and MN (red), Pax2 for dI4-V0/V1 (green) (T).

View larger version (60K): [in this window] [in a new window]   Fig. 2. Wnt patterning activity is mediated by the canonical pathway. (A) Activators of the canonical Wnt pathway (left) generate different transcriptional activities in vivo (centre). Embryos were electroporated with Wnt1/3a, β-cateninCA, TcfVP16 and Wnt4, together with the TopFlash reporter containing HMG-binding sites and normalization plasmid, and assayed 24 hours PE for luciferase activity. Graph shows normalized luciferase units. (B-G) Twenty-four hours PE, β-cateninCA caused expansion of Pax7 (B) and Pax6 (C) expression in those cells expressing GFP (D), and loss of ventral Olig2 (E) and Nkx2.2 (F) expression in a cell-autonomous way. (G) GFP expression on electroporated cells. (H) Expression of Wnt4 in a HH stage 18 embryo. (I-N) Twenty-four hours PE, Wnt4 produced wild-type expression of Pax7 (I) and Pax6 (J) in cells expressing GFP (K), and wild-type expression of Olig2 (L) and Nkx2.2 (M) in cells expressing GFP (N).

View larger version (64K): [in this window] [in a new window]   Fig. 3. Tcfs expressed throughout the DV axis regulate patterning of the neural tube. (A-D) HH stage 18 embryos showing expression of chick TCF1 (A), chick TCF3 (B), chick TCF4 (C) and chick LEF1 (D). (E) In vivo quantitative analysis of the repressors activities of components of the canonical Wnt pathway. Embryos were electroporated with mutant versions of Tcf proteins. Diagram shows schematic representation of DNAs. Graphics show normalized luciferase units. All mutant Tcfs abolished β-catenin-induced luciferase activity. (F-K) Eight hours PE of Tcf3DN, dorsal Pax7 (F) and Pax6 (G) showed wild-type expression. GFP as a reporter of transgene expression (H). Ectopic expression of ventral Olig2 (I) and Nkx2.2 (J) was induced in cells expressing the transgene (K). (L-U) Twenty-four hours PE of Tcf3DN, expression of Pax7 (L) and Pax6 (M) was lost in electroporated cells. GFP as a reporter of transgene expression (N). Intermediate genes Dbx1 (O) and Dbx2 (P), and ventral genes Olig2 (Q), Nkx2.2 (R), Foxa2 (S), Nkx6.2 (T) and Nkx6.1 (U) were all dorsally activated.

View larger version (58K): [in this window] [in a new window]   Fig. 4. Wnt activity in DV patterning is independent of Bmp but dependent on Shh/Gli activity. (A,B) HH stage 11/12 embryos were electroporated with Wnt1 and Wnt3a, and analysed 24 hours PE, for the expression of Bmp4 and Bmp7 by in situ hybridization. (A) Bmp4 expression was weakly expanded ventral to the roof plate. (B) Ectopic Bmp7 expression was induced on the electroporated side. (C) Electroporation of chick noggin resulted in the loss of Lhx2/9-expressing dI1. (D,E) Noggin electroporation alone did not modify dorsal expression of Pax7. (F) Co-electroporation of Wnt1/3a with noggin resulted in the expansion of Pax7 expression. (G,H) Noggin electroporation alone resulted in the wild-type expression of Olig2 and Nkx2.2. (I) Co-electroporation of Wnt1/3a with noggin resulted in the reduction of Olig2 (green) and Nkx2.2 (red) expression. (J-L) HH stage 11/12 embryos electroporated with Wnt1/3a were analysed 8 or 24 hours PE for the expression of Gli3 and Gli2 by in situ hybridization. (J) Eight hours PE of Wnt1/Wnt3a there was a moderate ventral expansion of Gli3 expression. (K) Twenty-four hours PE, over- and ectopic expression of Gli3 occurred. (L) Twenty-four hours PE there was overgrowth of the electroporated side without a change in expression of Gli2. (M-R) Wnt regulation of progenitor gene expression is dependent on Gli activity. (M) Twenty-four hours PE of GliZnF alone there was no modification of Pax7 expression (N,O) Co-electroporation of GliZnF abolished Wnt-induced ventral expansion of Pax7. (O) GFP as a reporter of transgene expression. (P) Twenty-four hours PE, GliZnF alone reduced Nkx2.2 (red) expression. (Q,R) Co-electroporation of Wnt1/3a and GliZnF resulted in a partial rescue of Olig2 (green) and Nkx2.2 (red) expression. (S) Quantitative analysis of Olig2+ and Nkx2.2+ cells on electroporated versus non-electroporated side of the spinal cord (24 hours PE of indicated DNAs). (T) In vivo quantitative analysis of the transcriptional activities for several components of the Wnt/β-catenin and the Shh/Gli pathways on a Tcf (TopFlash) transcriptional reporter. Embryos were electroporated with the indicated DNAs. Graph shows normalized luciferase units. GliZnF had no positive effect on the TopFlash reporter and cannot repress Wnt-induced luciferase. Electroporation of TcfHMG blocked all induced luciferase activity.

View larger version (58K): [in this window] [in a new window]   Fig. 5. Wnt signalling controls Gli3 expression in the dorsal spinal cord. (A-C) Embryos were electroporated with TcfDN and analysed for the expression of Gli3 and Gli2 by in situ hybridization. (A) Eight hours PE, TcfDN caused a reduction of Gli3. (B) Twenty-four hours PE, co-electroporation of TcfDN resulted in the loss of Gli3 expression. (C) Twenty-four hours PE, co-electroporation of TcfDN caused the reported growth arrest of the electroporated side, without changing expression of Gli2. (D-F) Analysis of mouse Gli3 expression in wild-type (Wnt1+/+; Wnt3a+/+), double heterozygous (Wnt1-/+; Wnt3a-/+) and double homozygous (Wnt1-/-; Wnt3a-/-) 10.5 dpc mouse embryos, by in situ hybridization. (G) Schematic representation of the human GLI3 locus. Conserved coding sequences are depicted in blue and conserved non-coding sequences in pink. Grey arrow indicates the length of the GLI3 gene and the direction of transcription. Tcf-binding sites are depicted in green. (H-R) Activity of HCNR1-R4 as putative enhancers tested by in ovo electroporation. (H) Embryos were co-electroporated with pCMV-DsRed as electroporation control, and analysed 24 hours later for GFP and RFP expression. (I,J) Embryos electroporated with the control empty vector showed only red expression. (K,L) Embryos electroporated with R1 showed only weak dorsal GFP expression. (M,N) HCNR2 electroporation resulted in strong dorsal GFP expression. (O,P) HCNR3 electroporation resulted in dorsal GFP expression, although weaker. (Q,R) HCNR4 electroporation resulted in only weak GFP expression. (S) In vivo quantitative analysis of the transcriptional activities of HCNR1-4. Embryos were electroporated with each of the amplified HCNRs (R1-R4) alone, or together with β-cateninCA or TcfDN. Embryos were all co-electroporated with a renilla-luciferase reporter construct for normalization, harvested after 24 hours of incubation and luciferase activity quantitated. Graph shows normalized luciferase units.

View larger version (14K): [in this window] [in a new window]   Fig. 6. The Wnt canonical pathway regulates transcriptional Shh/Gli activity. (A) In vivo quantitative analysis of the transcriptional activities of several components of the Wnt/β-catenin and the Shh/Gli pathways on a Gli-BS reporter. HH stage 11/12 embryos were electroporated with the indicated DNAs. Embryos were assayed 24 hours PE for luciferase activity. The mutant Gli3 protein, Gli3Act, showed high activity on the reporter; this is partially reduced by co-electroporation of Wnt1/Wnt3a. Wnt1/Wnt3a alone had no transcriptional effect on the Gli-BS reporter. Electroporation of TcfDN caused a potent activation of the reporter, activation that was inhibited by either wild-type or TCFHMG Gli3. (B) Schematic representation of a model for dorsoventral patterning of the spinal cord by antagonistic activities of Shh and Wnt signalling pathways.

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